Decades of research on the myriad of biological activities that the polyamines, putrescine, spermidine and spermine play in cellular processes have shown the profound role they play in life (Cohen, S. S., “A Guide to the Polyamines” 1998, Oxford University Press, New York). As polycations at physiological pH, they bind tightly to and strongly modulate the biological activities of all of the anionic cellular components.
Many stimuli involved in both normal and neoplastic growth activate the polyamine biosynthetic pathway. A great number of multidisciplinary studies have shown that the intracellular concentrations of the polyamines is highly regulated at many steps in their biosynthesis, catabolism and transport. The fact that cells contain such complex apparatus for the tight control of the levels of these molecules shows that only a very narrow concentration range is tolerated.
Polyamine transport into mammalian cells is energy and temperature dependent, saturable, carrier mediated and operates against a substantial concentration gradient (Seiler, N. et al. Polyamine transport in mammalian cells. Int. J. Biochem. 1990, 22, 211-218; Khan, N. A.; Quemener, V. et al. Characterization of polyamine transport pathways, in Neuropharmacology of Polyamines (Carter, C., ed), 1994, Academic, San Diego, pp. 37-60). Ample experimental proof exists that polyamine concentration homeostasis is mediated via this transport system. Changes in the requirements for polyamines in response to growth stimulation is reflected by increases in the transport activity. Stimulation of human fibroblasts to cell proliferation by serum or epidermal growth factor was followed by an 18-100 fold increase in the uptake of putrescine (DiPasquale, A. et al. Epidermal growth factor stimulates putrescine transport and omithine decarboxylase activity in cultures human fibroblasts. Exp. Cell Res. 1978, 116, 317-323; Pohjanpelto, P. Putrescine transport is greatly increased in human fibroblasts initiated to proliferate. J. Cell Biol. 1976, 68, 512-520). Tumors have been shown to have an increased rate of putrescine uptake (Volkow, N. et al. Labeled putrescine as a probe in brain tumors. Science, 1983, 221, 673-675; Moulinoux, J-P. et al. Biological significance of circulating polyamines in Oncology. Cell. Mol. Biol. 1991, 37, 773-783).
Inhibition of polyamine biosynthesis in cells in culture by α-difluoromethylomithine (DFMO), a well-studied mechanism-based inhibitor of ODC, causes a substantial depletion of intracellular putrescine and spermidine with resultant cell growth inhibition. Upon supplementing the culture media with exogenous polyamines this depletion causes transport activity to rise several-fold (Bogle, R. G. et al. Endothelial polyamine uptake: selective stimulation by L-arginine deprivation or polyamine depletion. Am. J. Physiol. 1994, 266, C776-C783; Alhonen-Hongisto, L. et al. Intracellular putrescine deprivation induces uptake of the natural polyamines and methylglyoxal bis (guanylhydrazone). Biochem. J. 1980, 192, 941-945). The cells then returned to their original rate of growth.
Genes for the polyamine transport protein or complex have been cloned from Escherichia coli and yeast (Kashiwagi, K. et al. J. Biol. Chem. 1990, 265, 20893-20897; Tomitori, H. et al. Identification of a gene for a polyamine transport protein in yeast. J. Biol. Chem. 1999, 274, 3265-3267). The genes for the mammalian transporter await identification. A subunit of the transporter from E. coli has been crystallized and its X-ray structure has been determined (Sugiyama, S. et al. Crystal structure of PotD, the primary receptor of the polyamine transport system in Escherichia Coli. J. Biol. Chem. 1996, 271, 9519-9525). This structure represents one of a few but growing number solved for spermidine-binding proteins. Since this structure was determined on a prokaryotic species its use in the design of mammalian transport inhibitors was deemed to be of limited value.
Several researchers have studied the ability of polyamine analogs to inhibit the uptake of 3H-spermidine into cells. Bergeron and coworkers studied the effect of addition of different alkyl group substitutions on the terminal nitrogen atoms of spermidine or spermine analogs (Bergeron, R. J. et al. Antiproliferative properties of polyamine analogs: a structure-activity study. J. Med. Chem. 1994, 37, 3464-3476). They showed that larger alkyl groups diminished the ability to prevent uptake of radiolabeled spermidine. They later concluded that increases in the number of methylenes between the nitrogen atoms decreased the ability to compete for 3H spermidine uptake (Bergeron, R. J. et al. A comparison of structure-activity relationships between spermidine and spermine antineoplastics. J. Med. Chem. 1997, 40, 1475-1494). They also concluded that the polyamine transport apparatus requires only three cationic centers for polyamine recognition and transport (Porter, C. W. et al. J. Cancer Res. 1984, 44, 126-128). Two groups have analyzed literature examples of the polyamine analogs' ability to inhibit 3H spermidine uptake into L1210 cells by CoMFA and QSAR methods (Li, Y. et al. Comparative molecular field analysis-based predictive model of structure-function relationships of polyamine transport inhibitors in L1210 cells. Cancer Res. 1997, 57, 234-239; Xia, C. Q. et al. QSAR analysis of polyamine transport inhibitors in L1210 cells. J. Drug Target. 1998, 6, 65-77).
A radiochemical assay is used for biochemical analysis of transport and has been used to study polyamine transport in yeast and a variety of mammalian cells (Kakinuma, Y et al., Biochem. Biophys. Res. Comm. 216:985-992, 1995; Seiler, N. et al., Int. J. Biochem. Cell Biol. 28:843-861, 1996). See, for example Huber, M. et al. Cancer Res. 55:934-943, 1995.
WO 99/03823 and its corresponding U.S. patent application Ser. No. 09/341,400, filed Jul. 6, 1999, (both of which are hereby incorporated in their entireties as if fully set forth) as well as the recent publications of Burns, M. R.; Carlson, C. L.; Vanderwerf, S. M.; Ziemer, J. R.; Weeks, R. S.; Cai, F.; Webb, H. K.; Graminski, G F. Amino acid/spermine conjugates: polyamine amides as potent spermidine uptake inhibitors. J. Med. Chem. 2001, 44, 3632-44 and Graminski, G. F.; Carlson, C. L.; Ziemer, J. R.; Cai, F., Vermeulen, N. M.; Vanderwerf, S. M.; Burns, M. R. Synthesis of bis-spermine dimers that are potent polyamine transport inhibitors. Bioorg. Med. Chem. Lett. 2002, 12, 35-40 describe some extremely potent polyamine transport inhibitors.
Citation of any reference herein is not intended as an admission that any of the foregoing is pertinent prior art, nor does it constitute any admission as to the contents or date of these documents.